Over 40 gram-positive pathogenic species from genera such as Streptococcus, Listeria, Brevibacterium, Clostridium, Bacillus, Arcanobacterium and Gardnerella express a member of the pore-forming cholesterol-dependent cytolysin (CDC) toxin family. Collectively, these pathogens represent a significant health burden worldwide and cause a wide variety of diseases, which include pneumonia, strep throat, earaches, wound infections and food-borne illnesses. The CDCs contribute to these diseases and are secreted by these pathogens as soluble monomeric proteins that bind mammalian host cells via membrane receptors, primarily cholesterol. These cell-bound monomers oligomerize into an oligomer complex comprised of 35-40 monomers, which then assembles and inserts a large ?-barrel pore into the plasma membrane of eukaryotic cells. Our studies of the assembly of the CDC pore complex have provided insight into the rational design of a new CDC-based vaccine and provided the first insights into the mechanism of mammalian immune defense proteins. Herein we will continue to investigate the molecular basis of cellular recognition and assembly of the large ?-barrel pore by the CDCs. In Aim 1, we will address the hypothesis that the structural differences in the membrane binding interface of the CDCs directs their binding to cholesterol that resides in specific domains on the cell wherein pore formation induces cellular effects that are unique to different CDCs. Understanding the basis of these differences will change the fundamental paradigms of how CDCs recognize and alter cellular function. In Aim 2 the fundamental features by which the CDCs control the assembly of the ?-barrel pore and its insertion to the membrane will be investigated. These studies will not only deepen our understanding of the CDC mechanism of pore formation, but will have relevance to a wide variety of bacterial and mammalian immune defense pore-forming proteins. In Aim 3, we will continue the investigation of the allosteric pathway by which the CDCs activate monomers upon cell binding to assemble into the giant CDC pore. Overall, the results of these studies will reveal new paradigms of CDC pore formation, provide a deeper understanding of how CDCs recognize and alter cellular function and impact the study of other pore-forming toxins and proteins.